BMC Genomics BioMed Central Research article Open Access A high-resolution radiation hybrid map of chicken chromosome 5 and comparison with human chromosomes Frédérique Pitel*1, Behnam Abasht2, Mireille Morisson1, Richard PMA Crooijmans3, Florence Vignoles1, Sophie Leroux1, Katia Feve1, Suzanne Bardes1, Denis Milan1, Sandrine Lagarrigue2, Martien AM Groenen3, Madeleine Douaire2 and Alain Vignal1 Address: 1Laboratoire de Génétique Cellulaire, INRA, Castanet-Tolosan, 31326, France, 2UMR Génétique Animale, INRA-ENSAR, Route de St Brieuc, Rennes, 35042, France and 3Animal Breeding and Genetics group, Wageningen University, Wageningen, 6709 PG, The Netherlands Email: Frédérique Pitel* - [email protected]; Behnam Abasht - [email protected]; Mireille Morisson - [email protected]; Richard PMA Crooijmans - [email protected]; Florence Vignoles - [email protected]; Sophie Leroux - [email protected]; Katia Feve - [email protected]; Suzanne Bardes - [email protected]; Denis Milan - [email protected]; Sandrine Lagarrigue - [email protected]; Martien AM Groenen - [email protected]; Madeleine Douaire - [email protected]; Alain Vignal - [email protected] * Corresponding author Published: 15 September 2004 Received: 19 May 2004 Accepted: 15 September 2004 BMC Genomics 2004, 5:66 doi:10.1186/1471-2164-5-66 This article is available from: http://www.biomedcentral.com/1471-2164/5/66 © 2004 Pitel et al; licensee BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The resolution of radiation hybrid (RH) maps is intermediate between that of the genetic and BAC (Bacterial Artificial Chromosome) contig maps. Moreover, once framework RH maps of a genome have been constructed, a quick location of markers by simple PCR on the RH panel is possible. The chicken ChickRH6 panel recently produced was used here to construct a high resolution RH map of chicken GGA5. To confirm the validity of the map and to provide valuable comparative mapping information, both markers from the genetic map and a high number of ESTs (Expressed Sequence Tags) were used. Finally, this RH map was used for testing the accuracy of the chicken genome assembly for chromosome 5. Results: A total of 169 markers (21 microsatellites and 148 ESTs) were typed on the ChickRH6 RH panel, of which 134 were assigned to GGA5. The final map is composed of 73 framework markers extending over a 1315.6 cR distance. The remaining 61 markers were placed alongside the framework markers within confidence intervals. Conclusion: The high resolution framework map obtained in this study has markers covering the entire chicken chromosome 5 and reveals the existence of a high number of rearrangements when compared to the human genome. Only two discrepancies were observed in relation to the sequence assembly recently reported for this chromosome. Background partly due to its position as a model species in various Chicken is the first major agricultural species for which fields of biology including embryo development, oncol- the complete genome sequencing was undertaken. This is ogy, immunology and evolution [1]. Moreover, as it is the Page 1 of 9 (page number not for citation purposes) BMC Genomics 2004, 5:66 http://www.biomedcentral.com/1471-2164/5/66 only bird species for which the genome study is so straints on the design of primers were to avoid presence of advanced, very much is expected from its use in compara- long introns, whose position and length was predicted on tive genome analyses for annotation, including that of the the basis of the orthologous human gene structure, and to human genome, by detection of conserved sequences design primers in the most divergent regions of the [2,3]. Its intermediate phylogenetic position between human/chicken alignment, to limit cross-amplification mammals and fishes will also certainly provide valuable with the hamster DNA present in the hybrids. One hun- information on the evolution of vertebrate karyotypes. dred and thirty nine primer pairs out of 156 (89.1%) ena- bled a successful amplification and the subsequent Radiation hybrid maps have a resolution power interme- mapping of the corresponding genes, confirming the high diate to that of the genetic and BAC contig maps and are success rate obtained when using the ICCARE software for also a powerful tool for the mapping of ESTs and genes by designing chicken PCR primers based on EST data [10]. simple PCR. They are thus useful at two levels: first, they can be used constructively as scaffolds for a correct Construction of the GGA5 RH map genome assembly or for detecting and correcting misas- Altogether, genotyping data was obtained for a total of sembled portions of the genome; second, before obtain- 169 markers, comprising 148 gene fragments (of which ing whole annotated genome sequences, they are very 139 developed using ICCARE) and 21 microsatellites efficient tools for inter-species comparative genome anal- from the GGA5 genetic map. Two-point analysis using a yses through the easy mapping of genes and ESTs [4-7]. LOD threshold of 6 enabled to constitute a group of 134 markers, including all the microsatellite markers from the The successful production of a RH panel in chicken is genetic map. The remaining 35 markers correspond to the quite recent [8], and therefore RH maps are only available external boundaries of the regions of conserved synteny for a limited number of chromosomes [9-11]. Having with human, from which ESTs were chosen for marker identified QTL (Quantitative Trait Loci) for fatness on development and map either to other chromosomes for chicken chromosome 5 [12], our objective was to build a which RH maps were developed (GGA1, 10, 18 or 24) or high-resolution and gene-rich RH map for this chromo- to unknown regions (data not shown). After multipoint some, as a basis for high precision comparative mapping analysis, a 1000:1 framework map 1315.6 cR6000 long, with human and for the development of new polymor- comprising a total of 73 markers including 12 microsatel- phic markers. lites and 61 ESTs was obtained. The remaining 61 markers are located relative to the framework map within confi- The available human/chicken comparative mapping data dence intervals, to build a comprehensive map (figure 1). indicated conservation of synteny between GGA5 and portions of HSA11, HSA14 and HSA15. In addition, two To compare the RH and the genetic maps, the best possi- genes from HSA1 had also been shown to be located on ble position of the non-framework common markers had GGA5 [13]. This information was used to develop mark- to be estimated. That of the markers on the RH map was ers from chicken EST sequence data orthologous to genes computed by the Carthagene program and is indicated in in these human regions, in addition to the existing mark- addition to the confidence interval. For the genetic map, ers from the chicken chromosome 5 genetic map. the central position of the marker's confidence interval was used as their most probable position. As a result, the While in the process of finishing our map, the first draft order of the markers on the RH map matches exactly that sequence assembly of the chicken genome was released of the same markers on genetic map [13], with only one (March 1st, 2004). The quality of both the GGA5 RH map notable discrepancy concerning the position of BRF1 (fig- and of the sequence assembly was therefore checked by ure 1). However, when the position of this gene was alignment of all the markers by BLAST searches. checked on the sequence assembly, the agreement was with the RH map, suggesting the position of this gene on Results and discussion the genetic map is erroneous. Development of EST markers In addition to the 21 microsatellite markers from the An average retention frequency of 21.4% was observed for genetic map, and 9 primer pairs chosen either from avail- the 134 GGA5 markers studied here, although with a high able primer data in the literature or designed using the variation, with values ranging from 6.8% to 55.7%. This gene sequence deposited in Genbank/EMBL, 156 primer finding is within the range observed in other studies pairs were chosen from chicken EST markers selected on reported on this panel: 21.9% overall retention using 42 the basis of the known conservations of synteny between markers chosen genome-wise [8], 24 % for GGA4 [11], human and chicken using the ICCARE (Interactive Com- 20.1 for GGA7 [10] and 18% for GGA15 [9]. As already parative Clustering and Annotation foR Est) software noticed for several species including human [14,15] or http://genopole.toulouse.inra.fr/bioinfo/Iccare/. Con- cow [16], but also for chicken chromosomes 4 and 7 Page 2 of 9 (page number not for citation purposes) BMC Genomics 2004, 5:66 http://www.biomedcentral.com/1471-2164/5/66 RH map (cR6000) Genetic map (cM) Retention rate (%/cR) 0 0.1 0.2 0.3 0.4 0.5 0.6 0 SPTB IGHMBP2 0 ABR0046 UNC93B1 AIP 0.0 DDB1 OSBP 10.0 MAX 17.3 LEI116 6 LEI116 RAD51 26.9 IVD 40.9 FLJ10634 55.0 NAV2 70.7 SLC17A6 86.2 GPR48 23 ACW0383 PAX6 111.5 28 MCW263 136.2 SPON1 PPFIBP2 162.9 ARNTL 200 CSTF3 32 LEI082 186.2 MCW263 210.6 AMPD3 34 ADL247 226.8 ADL247 243.7 ADL253 39 ACW0036 ST5 284.1 PSMA1 ADL253 303.0 SOX6 50 MCW193 323.7 RPS13 333.3 MYOD1 MCW090 PTPRJ